国际肿瘤学杂志

国际肿瘤学杂志 ›› 2015, Vol. 42 ›› Issue (1): 22-26.doi: 10.3760/cma.j.issn.1673-422X.2015.01.006

• 论著 • 上一篇    下一篇

靶向survivin基因微小RNA真核表达载体的构建及对人结肠癌细胞增殖的影响

崔运福, 郝涛, 王荣华, 李宝松, 马冲, 樊鹏   

  1. 256603山东省滨州医学院附属医院肛肠外科
  • 收稿日期:2014-08-18 修回日期:2014-10-08 出版日期:2015-01-08 发布日期:2015-01-07
  • 通讯作者: 郝涛 E-mail:anmly521@163.com

Construction and inhibitory effect of microRNA expression vector targeting survivin on proliferation of human colorectal carcinoma HT-29 cells

Cui Yunfu, Hao Tao, Wang Ronghua, Li Baosong, Ma Chong, Fan Peng   

  1. Department of Anorectal Surgery, Affiliated Hospital of Binzhou Medical University, Binzhou 256603, China
  • Received:2014-08-18 Revised:2014-10-08 Online:2015-01-08 Published:2015-01-07
  • Contact: Hao Tao E-mail:anmly521@163.com

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摘要： 目的 构建靶向survivin基因的微小RNA（miRNA）真核表达载体，探讨其对人结肠癌细胞（HT-29）增殖、凋亡的影响。方法设计合成针对survivin基因的miRNA序列，定向克隆至pcDNATM6.2GW/Em GFPmiR真核表达载体上，构建靶向survivin的miRNA重组质粒。HT29细胞接种于6孔板，分为空白对照组、转染试剂组、空质粒组（阴性对照组）以及目的基因组（阳性对照组）4组。瞬时转染后收集各组细胞，应用流式细胞仪检测各组细胞的增殖指数及凋亡率，实时荧光定量聚合酶链反应和免疫印迹法（Western blot）检测靶基因（survivin）mRNA和蛋白的表达。结果HT29细胞转染后，目的基因组增殖指数与正常组、转染试剂组、空质粒组相比明显降低（17.98%±2.35% vs 38.04%±2.11% vs 36.73%±2.51% vs 36.57%±3.05%; t=20.05，P<0.01；t=18.75，P<0.01；t=18.59，P<0.01），凋亡率明显增高（19.54%±1.74% vs 3.13%±0.29% vs 3.70%±0.44% vs 3.61%±0.50%; t=16.40，P<0.01；t=15.84，P<0.01；t=15.92，P＜0.01）。目的基因组survivin mRNA与其他各组相比明显降低（t=0.68，P<0.01；t=0.58，P<0.01；t=0.61，P<0.01），蛋白的表达亦明显降低（t=0.64，P<0.01；t=0.62，P<0.01；t=0.67，P＜0.01）。结论靶向沉默结肠癌HT-29细胞的survivin基因可以明显抑制细胞增殖，并诱导细胞凋亡。

关键词: 微RNAs, 结肠肿瘤, 细胞增殖, Survivin

Abstract: Objective To construct microRNA (miRNA) expression vector targeting surviving, and to investigate its effect on transfected human colorectal carcinoma (HT29) cell apoptosis and proliferation. MethodsmiRNA targeting survivin was synthesized and transfected HT29 cells by lipofectin. HT29 cells were cultured in the 6 orifices. The cultured cells were divided into control, liposome, negative control and positive control groups. Transient transfected cells were collected and the proliferation index and apoptosis rate of HT29 cells were detected by flow cytometry. The expressions of survivin mRNA and protein were detected by RTPCR and Western blot. ResultsThe proliferation index and apoptosis rate of the positive control group were significantly higher compared with normal group, transfection group and mockvehicle group (17.98%±2.35% vs 38.04%±2.11% vs 36.73%±2.51% vs 36.57%±3.05%; t=20.05, P<0.01; t=18.75, P<0.01; t=18.59, P<0.01; 19.54%±1.74% vs 3.13%±0.29% vs 3.70%±0.44% vs 3.61%±0.50%; t=16.40, P<0.01; t=15.84, P<0.01; t=15.92, P＜0.01). Survivin mRNA and protein expression levels were specifically suppressed in transfected HT29 cells (t=0.68, P<0.01; t=0.58, P<0.01; t=0.61, P<0.01; t=0.64, P<0.01; t=0.62, P<0.01; t=0.67, P＜0.01). ConclusionSurvivin targeted silence can effectively decrease the expression of survivin mRNA and protein, induce colorectal carcinoma HT29 cell apoptosis and inhibit cell proliferation.

Key words: MicroRNAs, Colonic neoplasms, Cell proliferation, Survivin